Chemistry Department, University of Nebraska, Lincoln, Nebraska 68588, United States.
Anal Chem. 2013 May 7;85(9):4453-60. doi: 10.1021/ac303734c. Epub 2013 Apr 17.
This report used high-performance affinity microcolumns to examine the changes in binding by sulfonylurea drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 μL of plasma or serum and 6-12 nmol of protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4-8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple drugs and experimental conditions over hundreds of sample application cycles. Both the overall binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in site-specific binding that occurred between sulfonylurea drugs or as the level of HSA glycation was varied. This method is not limited to clinical samples of glycated HSA but could be adapted for work with other modified proteins of interest in personalized medicine.
本报告使用高效亲和微柱研究了从糖尿病患者个体分离出的体内糖化 HSA 与磺酰脲类药物结合的变化。开发了一种免疫萃取方法，仅使用 20μL 血浆或血清和 6-12nmol 蛋白质，即可从临床样本中分离 HSA 和糖化 HSA 来制备每个亲和微柱。结果表明，亲和微柱可用于前沿分析或区带洗脱研究，每个运行通常仅需 4-8 分钟。微柱具有良好的稳定性，允许在数百个样品应用循环中获得多种药物和实验条件的数据。通过前沿分析测量的整体结合以及通过区带洗脱检查的特定部位相互作用，均与先前用类似修饰水平的体外糖化 HSA 获得的数据良好一致。还可以直接比较磺酰脲类药物之间或 HSA 糖化水平变化时发生的特定部位结合变化。该方法不仅限于临床糖化 HSA 样本，也可以适应个性化医学中其他感兴趣的修饰蛋白的研究。
